Acidic polycyclic ether antibiotic

ABSTRACT

An acidic polycyclic ether antibiotic, having structure established by X-ray crystallography, is formed by fermentation of a novel microorganism, Streptomyces sp. ATCC 53862. This novel antibiotic is useful as an anticoccidial in chickens, in the prevention or treatment of swine dysentery, and as a growth promotant in cattle and swine.

BACKGROUND OF THE INVENTION

The present invention concerns a new acidic polycyclic ether antibiotichaving the formula: ##STR1## wherein in Me=CH₃ and PR=CH₃ CH₂ CH₂,having absolute stereochemistry as shown; pharmaceutically acceptablecationic salts thereof; nutrient feed compositions comprising saidantibiotic for poultry, cattle or swine; its use as an anticoccidialagent in poultry, in the treatment or prevention of swine dysentery, oras a growth promotant in cattle or swine; a fermentation method for itspreparation; and the Streptomyces sp. microorganism which produces saidantibiotic in said fermentation method.

The compound (I), which is a homolog of monensin, is a new member of theacidic polycyclic ether group of antibiotics. This family includes, inaddition to monensin (The Merck Index, 10th Ed., Merck and Co., Inc.,Rahway, N.J., 1983, monograph no. 6100), such well known agents asnigericin (loc. cit., monograph no. 6390), narasin (loc. cit., monographno. 6271), lasalocid (loc. cit., monograph no. 5204), and salinomycin(loc. cit., monograph no. 8193). The subject has been reviewed byWestley, "Polyether Antibiotics", Adv. Appl. Microbiol., vol. 22, pp.177-223 (1977). These compounds are generally known as coccidiostats, asfeed additive-growth promotants, and/or as agents useful against swinedysentery.

SUMMARY OF THE INVENTION

A culture of Streptomyces sp., ATCC 53862, when fermented under aerobicconditions in aqueous media, produces a new acidic polycyclic etherantibiotic, a compound having the formula (I), as specified above.

The present invention is directed to said compound of the formula (I),including the pharmaceutically-acceptable cationic salts thereof, and toa process for its preparation which comprises fermentation of saidStreptomyces sp. ATCC 53862 in an aqueous nutrient medium comprising anassimilable source of carbon and nitrogen until a recoverable amount ofsaid compound of the formula (I) is formed, preferably under submergedaerobic conditions. For use as an anticoccidial agent, in the preventionor treatment of swine dysentery, and/or as a growth promotant, thecompound (I) is not necessarily separated from the fermentation andisolated in substantially pure form, but is alternatively used in crudeform, either in precipitated form admixed with mycelium (recovered byfiltration of the fermentation medium), or in solids obtained by spray-or freeze-drying the entire fermentation medium.

Said pharmaceutically-acceptable cationic salts include, but are notlimited to, those of sodium, potassium, calcium, ammonia,N,N'-dibenzylethylenediamine, N-methylglucamine (meglumine) anddiethanolamine. The preferred cationic salts are those of potassium andsodium.

The present invention is also directed to nutrient feed compositions,one for cattle or swine which comprises the compound of the formula (I)in an amount effective to promote growth and/or improve the feedutilization of said cattle or swine, or to prevent or treat dysentery inswine; and the other for poultry which comprises the compound of theformula (I) in an amount effective to control coccidial infection insaid poultry.

The present invention is further directed to a method for promotinggrowth and/or increasing the efficiency of feed utilization in swine orcattle which comprises administering to said swine or cattle a growthpromoting or feed-utilization efficiency promoting amount of thecompound of the formula (I), particularly in the form of a nutrient feedcomposition; to a method for preventing or treating dysentery in swinewhich comprises administering to said swine a compound of the formula(I) in an amount effective in preventing or treating said dysentery insaid swine; and to a method for controlling coccidial infections inpoultry which comprises administering to said poultry an anticoccidiallyeffective amount of the compound of the formula (I), particularly in theform of a nutrient feed composition.

Finally, the present invention is directed to a biologically pureculture of Streptomyces sp. ATCC 53862, said culture being capable ofproducing the compound of the formula (I) in a recoverable quantity uponfermentation in an aqueous nutrient medium comprising assimilablesources of carbon and nitrogen; including said culture in freeze-driedform.

DETAILED DESCRIPTION OF THE INVENTION

The culture capable of producing the present polycyclic ether antibioticof the formula (I) is designated Streptomyces sp., and has beendeposited under the Budapest Treaty in The American Type CultureCollection, Rockville, Md. as the type culture under their accessionnumber ATCC 53862. This culture will be irrevocably and withoutrestriction or condition released to the public upon issuance of apatent. Permanency of the deposit of this culture at The American TypeCulture Collection at Rockville, Md. and ready accessibility thereto bythe public are afforded throughout the effective life of the patent inthe event the patent is granted. Access to the culture is availableduring pendency of the application under 37 CFR 1.14 and 35 USC 122. Allrestrictions on the availability to the public of the culture depositedwill be irrevocably removed upon granting of the patent.

This novel culture was derived from a soil sample collected in Arnprior,Ontario, Canada and identified in the culture collection of Pfizer Inc.as N765-21. Its description and classification were provided by Dr. L.H. Huang. This culture was found to have the narrow hyphae of theActinomycetales, an aerial mycelium which produces spore chains, and anunfragmented substrate mycelium. The results of the whole cell analysesfurther indicate that it belongs to the genus Streptomyces.

A slant culture of the microorganism was planted into ATCC 172 broth andgrown for four days at 28° C. on a shaker. It was then centrifuged for20 minutes, washed three times with sterile distilled water, and plantedon media commonly used for identification of members of theActinomycetales.

The cultures were incubated at 28° C. and the results read at varyingtimes, but most commonly at fourteen days. The colors were described incommon terminology, but exact colors were determined by comparisons withcolor chips from The Color Harmony Manual, fourth edition. The methodsof whole-cell amino acid and sugar analyses are those described inBecker et al., Appl. Microbiol., vol. 12, pp. 421-423 (1964), andLechevalier, J. Lab. Clin. Med., Vol. 71, pp. 934-944 (1968),respectively. Streptomyces flocculus ATCC 25453 was used for purposes ofcomparison.

The culture was identified as follows:

Yeast Extract-Malt Extract Agar (ISP #2 medium, Difco)--Growth good,yellowish to yellowish brown (2 nc, 3 ic) with some white aerialmycelium; moderately raised, wrinkled; reverse yellowish (2 nc); solublepigment yellowish brown (3 lc).

Oatmeal Agar (ISP #3 medium, Difco)--Growth moderate to good, cream (2ca), with some white aerial mycelium; slightly raised, smooth; reversecream to pale yellowish (2 ca, 2 ea); soluble pigment cream (2 ca).

Inorganic Salts-Starch Agar (ISP #4 medium, Difco)--Growth good, whiteto yellowish (2 ga, 2 ic); thin to raised, smooth to slightly wrinkled,with white aerial mycelium; reverse yellowish (2 ga, 2 ic); solublepigment cream (2 ca).

Glycerol-Asparagine Agar (ISP #5 medium, Difco)--Growth poor tomoderate, cream (2 ca), thin, smooth, or appearing as isolated colonies;aerial mycelium sparse, white; reverse cream (2 ca); no soluble pigment.

Czapek-Sucrose Agar (Waksman, "The Actinomycetes", v. 2, medium #1, p.328, 1961)--Growth moderate, cream to pale yellowish (2 ca, 2 ea); thinto slightly raised, smooth, with white aerial mycelium; reverse same assurface; no soluble pigment.

Glucose-Asparagine Agar (ibid., medium #2)--Growth moderate to good,cream to yellowish (2 ca, 2 ga); slightly to moderate raised, smooth towrinkled; aerial mycelium none or sparse, white; reverse pale yellowish(2 ea); soluble pigment cream (2 ca).

Gordon and Smith's Tyrosine Agar (Gordon and Smith, J. Bacteriol.,69:147-150, 1955)--Growth moderate, cream (2 ca), thin to slightlyraised, smooth, no aerial mycelium; reverse pale yellowish (2 ea);soluble pigment yellowish (2 ga).

Casein Agar (Gordon and Smith, ibid.)--Growth moderate to good, cream (2ca), moderately raised, smooth to slightly wrinkled, no aerial mycelium;reverse pale yellowish (2 ea); with yellowish (2 ga) soluble pigment.

Bennett's Agar (Waksman, loc. cit., medium #30, p. 331)--Growth good,yellowish to dark yellowish (2 ic, 2 gc); slightly to moderate raised,smooth to wrinkled; aerial mycelium sparse, white; reverse yellowish (2ga, 2 ic); soluble pigment yellowish (2 ic).

Emerson's Aqar (ibid., medium #28, p. 331)--Growth good, cream,yellowish brown to brown (2 ca, 3 ic, 3 ie); moderately raised,wrinkled, no aerial mycelium; reverse yellowish (2 ga, 2 lc); solublepigment yellowish brown (3 lc).

Nutrient Aqar (ibid., medium #14, p. 330)--Growth moderate, cream (2ca), thin, smooth, no aerial mycelium; reverse cream to pale yellowish(2 ca, 2 ea); no soluble pigment.

Gelatin Agar (Gordon and Mihm, J. Bacteriol. 73, 15-27, 1957)--Growthgood, cream (2 ca), moderately raised, smooth but wrinkled toward edge,no aerial mycelium; reverse cream (2 ca); no soluble pigment.

Starch Agar (ibid.)--Growth good, cream to pale yellowish (2 ca, 2 ea),moderately raised, wrinkled, no aerial mycelium; reverse pale yellowish(2 ea); no soluble pigment.

Potato Carrot Agar (Lechevalier, Lab. Clin. Med., 71, 934-944, 1968, butuse only 30 g. potatoes, 2.5 g. carrots and 20 g. agar)--Growthmoderate, cream (2 ca) with some white aerial mycelium; thin, smooth toslightly granular; reverse colorless to cream (2 ca); no solublepigment.

Tap Water Agar (2%)--Growth poor to moderate, cream (2 ca), thin tosubmerged, smooth; aerial mycelium none to sparse, white; reversecolorless to cream (2 ca); no soluble pigment.

Morphological Properties--The morphological observations were made onISP Medium 9 (carbohydrate utilization medium) plus sucrose after 14days of incubation; spore mass in White color-series; spore chains inSection Rectiflexibles or Section Spirales, curved, hooked, looped or inopen coils of up to three turns, or arranged as an irregular mass at thetip; 10 to 30 spores per spore chain; sporophores monopodially branched;spores elliptical to cylindrical, 1.2-2.0(-2.2)×0.8-1.0(-1.1) microns;smooth, as revealed by scanning electron microscopy.

Biochemical Properties--Melanin not produced; hydrogen sulfide produced;gelatin liquefied; starch hydrolyzed; nitrate reduced to nitrite indextrose nitrate but not in organic nitrate; poor growth and nodecomposition on Jensen's cellulose broth; no growth and nodecomposition on Levine and Schoenlein's cellulose broth; coagulationand clearing on milk; casein digestion positive; tyrosine digestionnegative. Carbohydrate utilization: glucose, arabinose, fructose,mannitol, rhamnose, sucrose, xylose, inositol and raffinose allutilized.

    ______________________________________                                        Temperature Relations                                                         21° C.                                                                            28° C.                                                                            37° C.                                                                            45° C.                                ______________________________________                                        Excellent  Excellent  Good       No                                           Growth     Growth     Growth     Growth                                       ______________________________________                                    

Whole-Cell Analysis--The whole-cell hydrolysates containedLL-diaminopimelic acid, glucose and mannose.

The culture N765-21 is characterized by the white color of spores inmass, the negative melanin reaction, and the smooth spores which arecurved, hooked, looped or in an open coil of up to three turns. Thesubstrate mycelium was cream, pale yellowish to yellowish. Except forsome tints of cream, yellowish or yellowish brown, there were nodistinct soluble pigments produced. The culture utilized glucose,arabinose, fructose, inositol, mannitol, raffinose, rhamnose, sucroseand xylose. The whole-cell hydrolysates indicate the presence ofLL-diaminopimelic acid and the absence of diagnostic sugars.

Compared with the previously described species of Streptomyces, theculture N765-21 is related to S. pseudogriseolus, S. sclerotialus and S.flocculus. It differs from S. pseudogriseolus in its utilization ofsucrose and raffinose. It is different from S. sclerotialus in itsfailure to produce sclerotia and its opened rather than compact sporechains.

When the culture N765-21 and S. flocculus ATCC 25453 were comparedside-by-side, both resemble each other morphologically, culturally andbiochemically. However, the culture N765-21 differs from S. flocculus inthe positive starch hydrolysis, the failure to reduce organic nitrate,the poor rather than good growth on Jensen's cellulose broth, thefailure to peptonize milk, the failure to grow at 45° C., and thepositive rather than doubtful utilization of rhamnose.

On most of the media used, the culture N765-21 produced no or pooraerial mycelia, whereas S. flocculus produced abundant aerial mycelia.The substrate mycelium of the culture N765-21 was yellowish on Emerson'sagar, and pale yellowish on casein agar and tyrosine agar; but that ofS. flocculus was dark brown on Emerson's agar, yellow-brown on caseinagar, and brown on tyrosine agar. The soluble pigments produced by S.flocculus were darker on some media, e.g., dark brown on Emerson's agarand tyrosine agar, and yellow-brown on casein agar.

On the basis of the data presented above, the culture N765-21 isconsidered as a member of the genus Streptomyces and designatedStreptomyces sp. It has been deposited at the American Type CultureCollection under the accession number ATCC 53862.

The antibiotic compound (I) of the present invention is readily producedby the present Streptomyces sp. by growing at from about 24° to about36° C. under submerged conditions with agitation and aeration on mediaconsisting of carbohydrate sources such as sugars, starches, glycerol;organic nitrogen substances such as soybean meal, casamino acids, yeastextract; growth substances such as grain solubles, fish meal, cottonseed meal; mineral salts containing trace elements such as iron, cobalt,copper, zinc, etc. and calcium carbonate or phosphates as bufferingagents. After growth has been completed, the antibiotic is readilyrecovered by extracting the whole broth with an organic solvent such asn-butanol, methylisobutyl ketone, or chloroform at pH ranges from 4.0 to8.0; by filtering off the mycelium, which contains the precipitatedantibiotic, the filtrate being discarded; or by simply spray-drying orfreeze-drying the whole broth. Alternatively, the mycelium or the wholedried broth is extracted with one of said organic solvents. The purifiedantibiotic compound, if that is desired, is isolated from the organicextract by standard methods of concentration, salt or free acidformation, chromatography, precipitation and/or crystallization, asexemplified below.

In the usual manner of carrying out the fermentation, an inoculum isfirst prepared by scraping vegetative cells, growing on a suitablemedia, from slants or Roux bottles which have been inoculated withStreptomyces sp. ATCC 53862. The resulting vegetative cells are in turnused to inoculate shake flasks or inoculum tanks, also containingsuitable growth media. Alternatively, the inoculum tanks are inoculatedfrom the shake flasks. Following a suitable growth period (generally 120to 144 hours in shake flasks and 168 to 196 hours in inoculum tanks), afermenter, also containing suitable growth media, is inoculated underaseptic conditions with vegetative broth from the shake flasks orinoculum tanks. Upon completion of growth (generally about 120-196hours), the antibiotic compound is recovered in crude or pure form, asdesired, by one or another of the methods generally described above, orby specific methods which are exemplified below.

The compound of the formula (I) is tested for in vitro antibacterialactivity by standard methods in which the minimum inhibitoryconcentrations (MIC's) in mcg/ml against one or more microorganisms ismeasured. One such procedure is the one recommended by the InternationalCollaborative Study on Antibiotic Sensitivity Testing (Ericcson andSherris, Acta. Pathologica et Microbiologia Scandinav, Supp. 217,Section B: 64-68 [1971]), and employs brain heart infusion (BHI) agarand an inocula replicating device. Overnight growth tubes are diluted100 fold for use as the

standard inoculum (20,000-100,000 cells in approximately 0.002 ml. areplaced on the agar surface; 20 ml. of BHI agar/dish). Twelve 2 folddilutions of the test compound are employed, with initial concentrationof the test drug being 200 mcg/ml. Single colonies are disregarded whenreading plates after 18 hours at 37° C. The susceptibility (MIC) of thetest organism is accepted as the lowest concentration of compoundcapable of producing complete inhibition of growth as judged by thenaked eye. Like other polycyclic ether antibiotics, the present compoundof the formula (I) typically shows Gram positive antibacterial activity,as well as activity against Treponema hyodysenteriae, (the causativeagent of swine dysentery) as illustrated in Table (I).

                  TABLE I                                                         ______________________________________                                        IN VITRO ANTIBACTERIAL ACTIVITY OF THE                                        COMPOUND OF THE FORMULA (I)                                                   Organism          Strain No.                                                                              MIC, mcg/ml                                       ______________________________________                                        Clostridium perfringens                                                                         10A009    less than 0.39                                    Actinomyces pyogenes                                                                            14D002    less than 0.39                                    Treponema hyodysenteriae                                                                        94A007    less than 0.39                                    ______________________________________                                    

Efficacy data for the compound of the formula (I) and its salts againstcoccidial infections in chickens is obtained by the following method.Groups of 3-5 ten-day old pathogen free white leghorn cockerel chicksare fed a mash diet containing the compound (I) or its sodium and/orpotassium salt uniformly dispersed therein. After being on this rationfor 24 hours each chick is inoculated per os with oocysts of theparticular species of Eimeria being tested. Other groups of 3-5 ten-dayold chicks are fed a similar mash diet without compound (I) or itssalts. They are also infected after 24 hours and serve as infectedcontrols. Yet another group of 3-5 ten-day old chicks are fed the samemash diet without antibiotic and are not infected with coccidia. Theseserve as normal controls. The results of treatment are evaluated afterfive days in the case of E. acervulina, and six days for all otherchallenges.

The criteria used to measure anticoccidial activity consists of lesionscores of 0 to 4 for E. tenella after J. E. Lynch, "A New Method of thePrimary Evaluation of Anticoccidial Activity", Am. J. Vet. Res., 22,324-326, 1961; and 0 to 3 for the other species based on modification ofthe scoring system devised by J. Johnson and W. H. Reid, "AnticoccidialDrugs. Lesion Scoring Techniques in Battery and Floor Pen Experiments inChicks", Exp. Parasit., 28, 30-36, 1970. Activity is measured bydividing the lesion score of each treated group by the lesion score ofthe infected control. In this test, the compound (I) and its cationicsalts exhibit excellent activity against Eimeria tenella, E. acervulina,E. brunetti and E. necatrix infections in poultry when incorporated intothe mash diet of chickens at levels of about 20 to 100 ppm. For example,against a sensitive E. tenella, the compound of the formula (I) showed100% control of lesions at doses as low as 50 ppm.

The present compound of the formula (I) is also generally useful incombination with certain other known anticoccidial agents, such asnicarbazin, 4,4'-dinitrocarbanilide or a naphthalenamine, as defined byHamill et al., U.S. Pat. No. 4,582,822, cited above.

For the prevention or control of coccidiosis in poultry, the compound ofthis invention is orally administered to poultry in a suitable carrier.Conveniently, the medication is simply carried in the drinking water orin the poultry feed, so that a therapeutic dosage of the agent isingested with the daily water or poultry ration. The agent can bedirectly metered into drinking water, preferably in the form of a liquidconcentrate, or added directly to the feed as such, or in the form of apremix or concentrate. A premix or concentrate of therapeutic agent in acarrier is commonly employed for the inclusion of the agent in the feed.The therapeutic agent can be in substantially pure form (e.g., the freeacid, or a pharmaceutically-acceptable salt thereof), in assayed crudeform such as wet or dry mycelium or dried whole broth. Suitable carriersare liquid or solid, as desired, such as water, various meals; forexample, soybean oil meal, linseed oil meal, corncob meal, and mineralmixes such as are commonly employed in poultry feeds. A particularlyeffective carrier is the poultry feed itself; that is, a small portionof poultry feed. The carrier facilitates uniform distribution of theactive materials in the finished feed with which the premix is blended.This is important because only small proportions of the present potentagents are required. It is important that the compound be thoroughlyblended into the premix and, subsequently, the feed. In this respect,the agent may be dispersed or dissolved in a suitable oily vehicle suchas soybean oil, corn oil, cottonseed oil, and the like, or in a volatileorganic solvent and then blended with the carrier. It will beappreciated that the proportions of active material in the concentrateare capable of wide variation since the amount of agent in the finishedfeed may be adjusted by blending the appropriate proportion of premixwith the feed to obtain a desired level of therapeutic agent.

High potency concentrates are blended by the feed manufacturer withproteinaceous carrier such as soybean oil meal and other meals, asdescribed above, to produce concentrated supplements which are suitablefor direct feeding to poultry. In such instances, the poultry arepermitted to consume the usual diet. Alternatively, such concentratedsupplements are added directly to the poultry feed to product anutritionally balanced, finished feed containing a therapeuticallyeffective level of one or more of the compounds of this invention. Themixtures are thoroughly blended by standard procedures, such as in atwin shell blender, to ensure homogeneity.

For use in poultry, the use levels of the compound described herein willvary under different circumstances. Continuous low-level medication,during the growing period; that is, during the first 5 to 12 weeks forchickens, is an effective prophylatic measure. In the treatment ofestablished infections, higher levels may be necessary to overcome theinfection. The use level of the compound (I) in feed will generally bein the range of about 20 to 100 ppm, preferably in the range of about 30to 60 ppm. When administered in drinking water, the level which will bethat which will provide the same daily dose of medication factored bythe weight ratio of the average daily consumption of feed to the averagedaily consumption of water.

The activity of the compound of the formula (I) and its salts inpromotion growth and/or increasing the efficiency of food utilization inswine or cattle can be measured directly by feeding test groups ofanimals various levels of the compound (I) or a salt in feed.Alternatively, British Pat. Specification No. 1,197,826 details an invitro rumen method for the evaluation of antibiotics in feeds.

For use in the prevention or treatment of swine dysentery, or inpromoting growth and/or increasing the efficiency of feed utilization incattle or swine the compound of the formula (I) or a salt is preferablyadministered as a feed additive. The feeds prepared according to methodsfully analogous to those detailed above for the preparation of poultryfeed, with the same concern for producing feeds in which the therapeuticagent is uniformly dispersed. The use level of the compound (I) incattle or swine feed will generally be in the range of about 20 to 100ppm. In ruminants the compound of the formula (I) can also be orallyadministered in the form of a bolus which is retained in therumenoreticular sac, releasing the therapeutic agent at a substantiallyconstant rate over a prolonged period of time, e.g., 4-8 weeks,providing a dose equivalent to that of the above daily dose in feed,i.e.:

    ______________________________________                                        average daily dose                                                                            =                                                             in milligrams                                                                 (20 to 100)     × average daily feed                                    ppm                     consumption in Kg.                                    ______________________________________                                    

Exemplary of such a controlled release bolus is that of Cardinal, U.S.Pat. No. 4,601,893.

The present invention is illustrated by the following examples. However,it should e understood that the invention is not limited to the specificdetails of these examples.

EXAMPLE 1 Fermentation of Streptomyces sp. ATCC 53862 Isolation of theCompound (I) as the Sodium Salt

The Streptomyces sp. was initially grown by inoculating solid media onslants or Roux bottles with the ATTC 53862 culture, using ATTC mediumNo. 172, prepared and having composition as follows.

    ______________________________________                                                           Grams/liter                                                ______________________________________                                        Glucose              10                                                       Soluble Starch       20                                                       Yeast Extract        5                                                        Casein Enzymatic Hydrolysate                                                                       5                                                        Calcium Carbonate    1                                                        Distilled Water to 1000 ml;                                                                        20                                                       pH to 7.0 with KOH; Add Agar                                                  ______________________________________                                    

Meanwhile, shake flasks were prepared using one or the other of thefollowing media:

    ______________________________________                                                    Grams/                 Grams/                                     C'          liter    JDYTT         liter                                      ______________________________________                                        Cerelose    10       Cerelose      10                                         Soy Flour   10       Corn Starch   5                                          Corn        5        Corn Steep Liquor                                                                           5                                          Fermentation                                                                  Solids                                                                        Corn Starch 10       Casein Enzymatic                                                                            5                                                               Hydrolysate                                              Sodium Chloride                                                                           5        Cobalt Chloride                                                                             0.002                                      Cobalt Chloride                                                                           0.002    Calcium Carbonate                                                                           3                                          Calcium Carbonate                                                                         1                                                                 ______________________________________                                    

One hundred ml of medium was distributed into 300 ml shake flasks andsterilized at 120° C. and 15 p.s.i. for 30 minutes. After cooling, themedium was inoculated with a vegetative cell suspension scraped from theabove Streptomyces sp. slant culture. The flasks were shaken at 28° C.on a shaker having a displacement of 1.5 to 2.5 inches and 150 to 200cycles per minute (CPM) for five to seven days.

Meanwhile, 5 liter fermentation vessels were prepared containing 3liters of one of the above C' or JDYTT media or the following media:

    ______________________________________                                        UK1-2            Grams/liter                                                  ______________________________________                                        Cerelose         45                                                           Soy Flour        10                                                           Corn Steep Liquor                                                                              10                                                           Cobalt Chloride  0.002                                                        Magnesium Sulfate                                                                              0.10                                                         Calcium Carbonate                                                                              3                                                            Manganese Sulfate                                                                              0.10                                                         Ferric Sulfate   0.10                                                         ______________________________________                                    

An antifoaming agent (polypropylene glycol, P2000, containing 10%ethylene oxide by weight, 1 ml) was added, and the vessels were sealedand sterilized at 120° C. and 15 p.s.i. for 45 minutes. The cooledvessels were then inoculated with one shake flask (ca 3% inoculum),fermented for 120 to 168 hours at 30° C., stirring at 1700 revolutionsper minute (RPM) with an air rate of one volume of air per volume ofliquid per minute.

When the fermentation was completed (based on an antibiotic disc assayversus B. subtilis ATCC 6633) the fermenters were stopped and filteredat the natural pH with the aid of a diatomaceous earth. The filter cakewas slurried in methanol, concentrated in vacuo, diluted with 2-3volumes of water then extracted 2X with 1/3 to 2/3 volume of eithermethylisobutyl ketone or n-butanol. The solvent layer was separated fromthe aqueous phase by aspiration or centrifugation, sparkled andconcentrated in vacuo to yield the antibiotic of the formula (I) incrude form as a viscous oil.

The bioactivity of the broth and subsequent recovery streams wasfollowed by using a sensitive strain of Bacillus subtilis ATCC 6633 orStaphylococcus aureus ATCC 6538. The components in the broth andrecovery streams can be visualized by using Analtech silica gel GFplates employing ethyl acetate as eluant. The developed plates weresprayed with vanillin reagent (3 g vanillin in 75 ml ethanol and 25 ml85% phosphoric acid) and heated to 80° C. The antibiotic product of theformula (I) appears as an orange-brown spot. The developed tlc plate canalso be overlayed with agar seeded with either S. aureus or B. subtilisto which 2,3,5-triphenyl-2H-tetrazolium chloride monohydrate has beenadded and incubated at 37° C. for 16 hours to visualize the antibiotic(white spots against a pink background).

In a second fermentation, sixty jar fermenters containing approximately180 liters of fermented broth were recovered by extraction with 1/2volume of methylisobutyl ketone, separated by extraction andconcentration of the solvent, yield 31.6 g. The solid waschromatographed on silica gel gradiently eluted with chloroform followedby 0.5%, 1% and finally 2% CH₃ OH in CHCl₃. The activity was followed bytlc using silica gel plates developed with ethyl acetate and visualizingthe active cuts with vanillin reagent at 80° C. The ionophore appears asorange brown bonds. Concentration of the active cuts produced 6.9 g ofan oil, which was crystallized from warm diethyl ether to yield 2.62 gof crystalline compound (I). Reworking of the mother liquors yielded anadditional 0.55 g of the same product.

EXAMPLE 2 Scale-up Fermentation

Scale-up in large fermentation vessels was carried out by preparingshake flasks containing 0.7 liters of C' or JDYTT medium. The shakeflask inoculum was fermented for 5 to 7 days at 28° C., and used toinoculate a 6000 liter fermentation vessel containing 4000 liters ofJDYTT medium. Approximately one liter of inoculum was used in the tank.The fermentation, after proceeding for 7 to 10 days, was harvested.

The whole broth was extracted with 1600 liters of methylisobutyl ketoneat natural pH, and the layers separated on a DeLaval separator. Theorganic extract was concentrated under vacuum, first by vacuum pan andthen on a cyclone still and rotary evaporator to yield 8 liters of anoil. This oil was chromatographed on column grade silica gel slurried inhexane. The column was developed with ethyl acetate. Product containingcuts, identified by tlc using the method described above, were combined,stripped and the residue taken up in ethyl acetate, treated withactivated carbon, and filtered. The filtrate was shaken with dilute H₃PO₄ and then with dibasic sodium phosphate buffer to form the sodiumsalt, dried over Na₂ SO₄, concentrated, and 24.4 g of the compound ofthe formula (I) crystallized as the sodium salt by the addition ofheptane; tlc Rf 0.4 (10:1 CHCl₃ :CH₃ OH), 0.35 (ethyl acetate).

C-13 nmr [chemical shift (ppm) in CDCl₃ with number of hydrogens inparentheses]: 181.2* (0), 110.0 (0), 106.9 (0), 98.3* (0), 85.8* (1),85.3 (0), 85.2 (1), 83.0* (1), 82.6 (1), 76.4* (1), 74.4 (1), 70.6 (1),68.3* (1), 64.9* (2), 57.9 (3), 56.9 (3), 45.0* (1), 40.6 (2), 39.2*(2), 37.4 (1), 36.5* (1), 35.6 (2), 34.8* (1), 34.4 (1), 33.5* (2),33.2* (2), 31.8* (1), 30.0 (2), 27.4* (3), 27.2 (2), 17.0 (2), 16.8*(3), 16.0* (3), 14.7 (3), 14.5* (3), 11.0* (3), and 10.5* (3).

*Monensin shows identical chemical shifts, and in addition peaks at107.0 (0), 85.2 (0), 84.9 (1), 82.5 (1), 74.5 (1), 70.4 (1), 57.8 (3),37.5 (1), 35.7 (2), 34.3 (1), 29.8 (2), 27.3 (2) and 8.1 (3).

The structure of the compound of the formula (I) was proven by X-raycrystallographic analysis.

We claim:
 1. A process for the preparation of a compound of the formula ##STR2## wherein Me is CH₃ and Pr is CH₃ CH₂ CH₂, or a pharmaceutically acceptable salt thereof, which comprises fermentation of Streptomyces sp. ATTC 53862 under submerged aerobic conditions in an aqueous nutrient medium comprising an assimilable source of carbon and nitrogen until a recoverable amount of said compound is formed.
 2. A process of claim 1 wherein said compound is separated from the fermentation medium.
 3. A process of claim 1 wherein said compound in precipitated form is recovered as a mixture comprising mycelium by filtration of the fermentation medium.
 4. A process of claim 1 wherein said compound is recovered in crude form by spray- or freeze-drying the entire fermentation medium.
 5. A biologically pure culture of Streptomyces sp. ATCC 53862, said culture being capable of producing a compound of the formula ##STR3## wherein Me is CH₃ and Pr is CH₃ CH₂ CH₂, in a recoverable quantity upon fermentation in an aqueous nutrient medium comprising assimilable sources of carbon and nitrogen.
 6. The culture of claim 5 in freeze-dried form. 